Antibacterial composition and its use in treating bacterial infections

ABSTRACT

An antibacterial composition that comprises as active compound at least one light isotope of an element selected from the group which consists of  1 H,  12 C,  16 O,  14 N,  39 K,  24 Mg,  64 Zn,  85 Rb,  28 Si,  54 Fe,  92 Mo,  74 Se,  58 Ni,  70 Ge,  52 Cr,  63 Cu,  50 V, or combinations thereof, wherein the composition is enriched for the at least one light isotope. A method of treating and preventing bacterial diseases in humans and non-human animals by administering the composition. The use of the said composition in human and veterinary medicine for the prevention and treatment of diseases in humans and non-human animals and also as an antiseptic and disinfectant.

FIELD OF THE INVENTION

The present invention relates to a novel antibacterial composition thatis enriched for one or more light isotopes of one or more chemicalelements as an active ingredient and the use of such a composition inthe treatment and prevention of bacterial infections in humans andnon-human animals.

BACKGROUND OF THE INVENTION

The variability of isotopes is known as the isotope effect, a termdescribing the mass-dependent variations of natural isotope contents fora particular element. The isotope effect is a consequence of theHeisenberg uncertainty principle on 75 levels of the energy distributionof molecular vibrations (Metallomics, 2016, Accepted Manuscript DOI:10.1039/C6MT00148C). It is known that the isotopic weight has an effecton the value of the effective radius of electron orbits of atoms andleads to changes in the characteristics of the fine structure of atomicenergy levels. Biochemical processes of organisms are highly dependenton the conditions of their occurrence, usually using resonant effects,so the slightest deformations of electron orbitals can lead todisruption of biochemical reactions.

A number of studies have demonstrated that the isotopic composition oftissues and organs can serve as a diagnostic marker. In particular, thestudy of correlations of Cu and Zn isotopes in blood showed theirpromising relationship to age, sex and pathologies. For example,assessment of the ratio of Cu isotopes in the blood serum is a newapproach to the diagnosis and prognosis of liver cirrhosis (see M.Costas-Rodriguez et al., Isotopic analysis of Cu in blood serum bymulti-collector ICP-mass spectrometry: a new approach for the diagnosisand prognosis of liver cirrhosis? Metallomics 7: 491-498 (2015)), andthe isotopic composition of Zn in breast tissue makes it possible todiagnose cancer (F. Lamer et al., Zinc isotopic compositions of breastcancer tissue, Metallomics 7: 107-112 (2015)).

In particular, it was found that natural water and most foods that areused by humans contain heavy isotopes of chemical elements. Every human,being a complex biochemical system, fractionates heavy isotopes duringhis lifetime. As a result, heavy isotopes, which accumulate in the humanorganism starting at birth, gradually “integrate” into the cells.

While not wishing to be bound by theory, the present inventors believethat each of hydrogen, carbon, oxygen, nitrogen potassium, magnesium,zinc, rubidium, silicon, iron, molybdenum, selenium, nickel, germanium,chromium, copper, and vanadium play important roles in autocatalyticreactions in the body of an animal, such as a human or other mammal. Theproducts of such autocatalytic reactions, such as proteins, playimportant chemical and structural roles in the body, including immunefunction. Fully functional products of such reactions require aspecific, “correct” chirality at various chiral centers within theproduct. The inventors further understand that heavy isotopes accumulatein the body beginning at birth such that, over time, the relativeabundance of each element's isotopes drifts further and further from thenaturally occurring relative abundance, becoming increasinglyover-weighted with respect to heavy isotopes. Heavy isotopes can affectautocatalytic reactions by reducing the proportion of products that havethe “correct” chirality. See, e.g., Tsuneomi Kawasaki et al., AsymmetricAutocatalysis Triggered by Carbon Isotope (¹³ C/ ¹² C) Chirality,Science 324: 492-95 (2009). This causes a reduction in the proportion ofproducts of autocatalytic reactions that are fully functional. In sum,the cumulative divergence of the body's isotope relative abundances fromthe natural relative abundance causes a decrease in the functionality ofvarious proteins and other molecules in the body, leading to a declinein health with age.

The present inventors believe such a decline can be countered byrestoring the body's original isotope relative abundances, or by movingthe isotope relative abundances in that direction. Similarly, pathogenicinfectious bacteria can be suppressed by treating them with lightisotopes of the elements listed above, which can alter the chirality ofthe autocatalytic products of such bacteria, resulting in their death orsuppressed growth. Thus, treatment with light isotopes can have the dualresult of improving the body's ability to fight off a bacterialinfection and simultaneously killing or suppressing the growth ofinfective bacteria. Further, the quantity of light isotope that iseffective may be proportional to the quantity of the correspondingelement that is present in the body. Where the body contains arelatively large quantity of the element, a correspondingly relativelylarge amount of the element's light isotope will be required to providean effective dosage amount. On the other hand, where the body contains arelatively small quantity of the element, a correspondingly relativelysmall amount of the element's light isotope will be required to providean effective dosage amount.

Light isotopes have been used in medicine, veterinary medicine, foodindustry and agriculture without producing adverse effects on organisms.

Patent RU2498807 purports to disclose a new treatment of acute radiationsickness which uses water with light isotopes as a therapeutic agent.The remedy is said to improve survival and accelerate recovery ofhematopoiesis and body weight.

International publication no. WO 01/82871 describes a method ofdiagnosis and treatment of colon cancer. This method uses zinc and theunstable isotope ⁶²Zn in the form of zinc acetate, zinc chloride andzinc sulfate, as well as the phosphate carrier.

According to U.S. publication no. 2016/0151415, a pharmaceuticalcomposition for improving health condition and treatment of pathologiesand degenerative diseases includes a pharmaceutically acceptable carrierand an active isotope selective ingredient that includes at least onechemical element wherein the isotope distribution is different from thatoccurring in nature, inherent in such chemical element. Thus ³⁹K, ²⁴Mg,⁶⁴Zn, ⁸⁵Rb, ²⁶Si, ⁴⁰Ca, ⁶³Cu, ⁵⁴Fe, ⁵²Cr, ⁵⁸Ni, ⁹²Mo, ¹⁰⁷Ag, ⁷⁹Br, ³⁵Cland combinations thereof are used as possible selective isotopes.However, the antibacterial effect of the above isotopes is not describedin the said application.

Publication No. GB2531207 purports to disclose an antibacterial agentwhich consists of at least one of the isotopes of hydrogen selected fromthe group including ¹H, ²H, ³H, ⁴H, ⁵H, ⁶H and ⁷H, a hydrogen molecule(H2), metal hydride, a hydrogen ion (H⁺), a hydride ion (H⁻) and atomichydrogen. The composition described in this publication is said toexhibit antibacterial activity and reduce propagation of drug-resistantmicroorganisms. In addition, hydrogen, after its exposure to pathogenicmicroorganisms, combines with oxygen to form water. According to thepublication, this eliminates adverse effects of the antibacterial agenton the organism to which it is administered, and the said agent haslittle effect on the host organism even if it is administered incombination with another drug(s).

There remains a need for new, effective antibacterial compositions.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides an antibacterial compositionthat comprises as an antibacterial agent at least one light isotopeselected from the group consisting of ¹H, ¹²C, ¹⁶O, ¹⁴N, ³⁹K, ²⁴Mg,⁶⁴Zn, ⁸⁵Rb, ²⁸Si, ⁵⁴Fe, ⁹²Mo, ⁷⁴Se, ⁵⁸Ni, ⁷⁰Ge, ⁵²Cr, ⁶³Cu, and ⁵⁰V,either in elemental form or in the form of a pharmaceutically acceptablesalt, compound, or complex, wherein the composition is enriched for theat least one light isotope relative to the natural abundance of theisotope. The at least one light isotope that the composition is enrichedfor preferably is present in a bacteriostatic or bactericidal effectiveamount. In preferred embodiments, the composition is suitable forvarious routes of administration, such as topical or oraladministration. In certain embodiments, the composition furthercomprises at least one additional ingredient suitable to the form of thecomposition, including carriers and excipients such as diluents,solvents (such as water), binders, lubricants, coloring agents, andpreservatives, which are conventional and known to the person ofordinary skill in the art. The composition preferably is formulated fora specific route of administration such as, but not limited to,injection (e.g. intravenous, intraperitoneal, or subcutaenousinjection), topical administration and oral administration. Specificexemplary forms of the composition include a topical solution, spray,lotion, salve, ointment, gel, cream, soap, shampoo, patch, powder andfoam, and an oral tablet, capsule, syrup, suspension, lozenge, gum,spray, and solution, and a solution or other composition suitable forintravenous, intraperitoneal, subcutaneous, or other route ofadministration by injection. Oral compositions of the invention may beformulated for immediate, delayed, or sustained release and may alsoformulated for enteric release. Topical compositions of the inventionpreferably include at least one absorption-enhancing agent such as DMSO.In a preferred embodiment, the at least one light isotope that thecomposition is enriched for comprises ⁶⁴Zn. In such an embodiment, the⁶⁴Zn preferably constitutes between about 90% and about 99.9% of thezinc in the composition. In alternative embodiments, any of the aboveantibacterial compositions can comprise as an antibacterial agent atleast one light isotope selected from any subgroup selected from thegroup consisting of ¹H, ¹²C, ¹⁶O, ¹⁴N, ³⁹K, ²⁴Mg, ⁶⁴Zn, ⁸⁵Rb, ²⁸Si,⁵⁴Fe, ⁹²Mo, ⁷⁴Se, ⁵⁸Ni, ⁷⁰Ge, ⁵²Cr, ⁶³Cu, and ⁵⁰V, either in elementalform or in the form of a pharmaceutically acceptable salt, compound, orcomplex, wherein the composition is enriched for the at least one lightisotope relative to the natural abundance of the isotope.

In various embodiments, the light isotope in the composition of theinvention is present in elemental form or in the form of one or more ofan oxide, sulfate, citrate, gluconate, chelate, or other compound, or inany other pharmaceutically acceptable form. The at least one lightisotope may be present in the composition in the form of a salt with apharmaceutically acceptable inorganic or organic acid. Exemplary saltsof the light isotope include sulfate, glutamate, asparaginate,aspartate, citrate, and ethylene diamine disuccinic acid (referred to inthis application both as “EDDA” and as “EDDS”) salts. An exemplary oxideis deuterium-depleted water.

In an embodiment, the composition of the invention further comprises anadditional antibacterial agent or other active ingredient. In anembodiment, the composition of the invention comprises an agent thatenhances the stability of the composition.

The light isotope may constitute between about 0.1% and about 99% of thecomposition by weight. When the light isotope is present in the form ofa salt, the anionic portion of the salt acts as a carrier. When water ispart of the said composition, it may function as a carrier and diluent.

The antibacterial composition, in accordance with the invention, can beused in human or veterinary medicine to treat infections in humans andnon-human animals, including veterinary mammals, caused by bacterialpathogens.

The invention also provides a method of preparing a composition of theinvention, such as a composition for administration orally, topically,or by injection, such as the compositions listed above, which comprisescombining a compound, complex, or pharmaceutically acceptable saltenriched for at least one of the above-listed isotopes with at least oneexcipient. The invention also provides a method of preparing acomposition of the invention which comprises incorporating an effectiveamount of a compound, complex, or pharmaceutically acceptable saltenriched for at least one of the above-listed isotopes into acomposition for administration to a human or veterinary animal, such asa veterinary mammal, such as a composition for administration orally,topically, or by injection, such as the compositions listed above. Inone embodiment, the method comprises combining a prepared topicalformulation or liquid oral formulation, such as an ointment, cream,lotion, gel, salve, spray, or solution, with an effective amount of acompound, complex, or pharmaceutically acceptable salt enriched for atleast one of the above-listed isotopes to provide a composition of theinvention.

The invention also provides a method of treating conditionscharacterized by bacterial infection or growth, such as bacterialinfections, in humans and veterinary animals, such as non-human mammals,comprising administering to a human or veterinary animal, e.g. aveterinary mammal, in need of such treatment (e.g. one that exhibitssuch a condition) an effective therapeutic amount of the composition ofthe invention. The invention also provides a method of preventingbacterial infections, and conditions characterized by bacterialinfection or growth, in humans and veterinary animals (such asveterinary mammals) in need of such treatment, such as humans andveterinary animals (such as veterinary mammals) known to be susceptibleto, prone to or highly susceptible to such infections or conditions,comprising administering to a human or veterinary animal (such as aveterinary mammal) in need of such treatment an effective prophylacticamount of the composition of the invention. In preferred embodiments ofsuch methods, the composition comprises a bacteriostatic or bactericidaleffective amount of ⁶⁴Zn_(e) (zinc that is enriched for zinc-64),present as an element or in the form of a pharmaceutically acceptablesalt, compound or complex thereof. In particularly preferredembodiments, ⁶⁴Zn constitutes between about 90% and about 99.9% of the⁶⁴Zn_(e).

The composition of the invention can also be used as a disinfectant ofsurfaces and as an antibacterial component in compositions used inagriculture. The invention thus also provides a method of disinfectingsurfaces comprising administering an effective amount of the compositionto the surface to be disinfected. In these aspects, the composition ofthe invention may advantageously be in the form of a sprayable liquid orpowder or a spreadable liquid or powder. The composition may be providedin concentrated form for later dilution with an appropriate vehicle orcarrier prior to use.

In another embodiment, the invention provides a composition for use inthe treatment or prevention of a bacterial infection, or of a conditioncharacterized by bacterial infection or growth, wherein the compositioncomprises as an antibacterial agent at least one light isotope selectedfrom the group consisting of ¹H, ¹²C, ¹⁶O, ¹⁴N, ³⁹K, ²⁴Mg, ⁶⁴Zn, ⁸⁵Rb,²⁸Si, ⁵⁴Fe, ⁹²Mo, ⁷⁴Se, ⁵⁸Ni, ⁷⁰Ge, ⁵²Cr, ⁶³Cu, and ⁵⁰V, either inelemental form or in the form of a pharmaceutically acceptable salt,compound, or complex, wherein the composition is enriched for the atleast one light isotope relative to the natural abundance of theisotope. The composition is as described in the preceding paragraphs. Ina preferred embodiment, the at least one light isotope that thecomposition is enriched for is ⁶⁴Zn_(e). More preferably, in such acomposition, ⁶⁴Zn constitutes between about 90% and about 99.9% of the⁶⁴Zn_(e). For example, ⁶⁴Zn may constitute at least about 95% of the⁶⁴Zn_(e) or at least about 99% of the ⁶⁴Zn_(e), such as about 99% or99.9% of the ⁶⁴Zn_(e).

The technical problem that is solved by the use of this inventionconsists in the development of an antibacterial composition which, onthe one hand, has high bacteriostatic and bactericidal activities and,on the other hand, does not cause any toxic effects associated with theuse of antibiotic active compounds and also does not cause thedevelopment of resistant microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

The advantages of the present invention will be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings wherein:

FIG. 1 presents diagrams (FIGS. 1a-1c ) that compare the MICs ofantibacterial compositions that contain zinc enriched for ⁶⁴Zn in theform of different salts: ⁶⁴Zn_(e) citrate, ⁶⁴Zn_(e) EDDA, ⁶⁴Zn_(e)sulfate, ⁶⁴Zn_(e) aspartate, ⁶⁴Zn_(e) glutamate.

FIG. 2 presents diagrams (FIGS. 2a-2c ) that compare the MBCs ofantibacterial compositions that contain zinc enriched for ⁶⁴Zn in theform of different salts: ⁶⁴Zn_(e) citrate, ⁶⁴Zn_(e) EDDA, ⁶⁴Zn_(e)sulfate, ⁶⁴Zn_(e) aspartate, ⁶⁴Zn_(e) glutamate.

FIG. 3 presents diagrams (FIGS. 3a-3c ) that compare the MIC ofantibacterial compositions of the invention that contain zinc enrichedfor ⁶⁴Zn in the form of the EDDA and sulfate salts to compositions thatcontain naturally-occurring zinc (not enriched for ⁶⁴Zn).

FIG. 4 presents diagrams (FIGS. 4a-4c ) that compare the MBC ofantibacterial compositions of the invention that contain zinc enrichedfor ⁶⁴Zn in the form of the EDDA and sulfate salts to compositions thatcontain naturally-occurring zinc (not enriched for ⁶⁴Zn).

FIG. 5 presents diagrams (FIGS. 5a-5c ) that compare antibacterialcompositions of the invention that contain zinc enriched for ⁶⁴Zn in theform of different salts to compositions that contain naturally-occurringzinc (not enriched for ⁶⁴Zn) and to known antibacterial agents.

DETAILED DESCRIPTION OF THE INVENTION

Definitions of the terms used in this application are given hereinafterto ensure their unambiguous understanding by specialists. Furthermore,unless otherwise specifically stated, all scientific and technical termsused herein have the same meaning as commonly understood by a person ofordinary skill in the art.

The term “isotope”, as used herein, refers to a variant of a particularchemical element which are rather similar in their physical and chemicalproperties but have a different atomic mass. According to theproton-neutron model developed by D. I. Ivanenko and W. Heisenberg(1932), atoms of all chemical elements consist of three types ofelementary particles: positively charged protons, negatively chargedelectrons, and neutrons that have no charge. The number of protons p inthe nucleus determines the atomic number Z of the chemical element inMendeleev's periodic table. The proton and the neutron, which have acommon name—nucleons—have almost identical weight. The mass of theneutron (1.00866 amu) is somewhat greater than the proton mass (1.00727amu). The electron mass is much smaller than that of the nucleons (forexample, the proton-to-electron mass ratio is 1836.13). Therefore, themass of the atom is concentrated in its nucleus. Hence, the mass numberof the atom A is connected with the atomic number by a simple relationA=p+n=Z+n, where n is the number of neutrons in the nucleus of an atom.The number of protons in the nucleus of an atom uniquely determines theposition of an element in the periodic table of the elements.Furthermore, the number of protons determines the number of electronspresent in a neutral atom thus determining the chemical properties ofthis atom. However, atoms with the same atomic number Z (and hence thenumber of protons p) may have different neutron numbers n. Thus atomswith different atomic mass numbers may occupy the same position on theperiodic table. Chemical elements having the same atomic number but adifferent atomic mass are known as isotopes.

As used herein, the term “light isotopes” refers to the followingisotopes: ¹H, ¹²C, ¹⁶O, ¹⁴N, ³⁹K, ²⁴Mg, ⁶⁴Zn, ⁸⁵Rb, ²⁸Si, ⁵⁴Fe, ⁹²Mo,⁷⁴Se, ⁵⁸Ni, ⁷⁰Ge, ⁵²Cr, ⁶³Cu, and ⁵⁰V.

The “natural abundance” of an isotope refers to the fraction of thetotal amount of the corresponding element that the isotope represents,on a mole-fraction basis (that is, not, for example, on a mass basis).For example, if ⁶⁴Zn had an earth natural abundance of 48.63%, thatwould mean that 48.63% of Zn atoms on earth are the isotope ⁶⁴Zn. When acomposition is “enriched” for a certain isotope, the abundance of theisotope in the composition is greater than the isotope's naturalabundance. For the preceding ⁶⁴Zn example, a composition in which ⁶⁴Znconstitutes more than 48.63% of the total Zn in the composition, on amole-fraction basis, would be “enriched” for ⁶⁴Zn. Throughout thisapplication, a subscript “e” following a light isotope chemical symbolor element name indicates that the designated element is enriched forthat isotope. For example, ⁶⁴Zn refers to the light isotope zinc-64,whereas ⁶⁴Zn_(e) refers to zinc that is enriched for zinc-64. Thus,“⁶⁴Zn_(e) aspartate,” for example, refers to zinc aspartate in which thezinc is enriched for zinc-64.

The proportion of an element that is present as a particular isotope ofthe element is often expressed relative to a ratio called the standardisotope ratio or SIR. The abundance of the isotope of interest is thenumerator of the SIR and the abundance of the most abundant isotope isthe denominator. For example, ¹²C is the most abundant carbon isotopeand ¹³C is a second carbon isotope. Assuming a standard abundance valuefor C-12 of 98.89% and a standard abundance value for ¹³C of 1.11%, theSIR for ¹³C would be 1.11/98.89=0.01122. Each SIR is obtained from areference material. Deviations from the SIR may be observed innon-reference materials.

For ease and convenience, the abundance of a heavy isotope in a materialof interest may be expressed relative to the heavy isotope's “standard”abundance in the reference material by reference to the difference inisotope ratios, expressed in parts per thousand or “‰” and referred toas delta−[isotope] or δ−[X], where “[X]” represents the isotope ofinterest. The δ value is calculated as ((R_(sample)−SIR)/SIR)×1000‰,equivalent to ((R_(sample)/SIR)−1)×1000‰, where R_(sample) is theisotope ratio of the sample under evaluation. For example, if the carbonstandard contains 99% ¹²C and 1% ¹³C, and the sample has 98.95% ¹²C and1.05% ¹³C, then the corresponding SIR, or ¹³C/¹²C of the standard, is1/99, or 0.0101, and the ¹³C/¹²C of the sample is 1.05/98.95, or 0.0106,so δ¹³C_(sample)=((0.0106/0.0101)−1)×1000‰=49.5‰ (also known as 49.5permil) or 0.0495.

Relative abundance of an isotope can also be expressed with respect todifferent isotopes' absolute abundances expressed in terms of “atompercent” and “fractional abundance.” Atom percent is calculated as(^(A)X/(sum of all X isotopes))×100, whereas fractional abundance issimply ^(A)X/(sum of all X isotopes), where “^(A)X” is a measure of thequantity of isotope A of element X in a sample, and “sum of all Xisotopes” is a measure of the total quantity of element X in a sample.Enrichment for a specific isotope in a sample of interest may beexpressed as a percentage of the fractional abundance or atom percent ofa reference standard. For example, if a reference standard containedpotassium, of which 93.3% was ³⁹K, then the atom percent of ³⁹K would be93.3% and its fractional abundance would be 0.933. If a sample were tocontain potassium, of which 95.0% was ³⁹K, then the sample would beenriched with respect to ³⁹K by (95.0−93.3)/93.3=1.82%. If a sample weresaid to be enriched with respect to ³⁹K by 5% relative to the standard,then the percentage of the potassium in the sample that is ³⁹K would be1.05×93.3=97.97%.

The degree of enrichment of a certain isotope also may be expressed withrespect to the difference D(I) (where “I” represents the identity of theisotope) between 100% and the isotope's natural abundance, expressed asa mole percentage of the total amount of the corresponding element. Forexample, if ⁶⁴Zn had a natural abundance of 48.63%, thenD(⁶⁴Zn)=100%−48.63%=51.37%. A sample's enrichment may then be expressedas the amount by which D is reduced. For the ⁶⁴Zn example, for a samplein which D(⁶⁴Zn) is reduced by 10%, D(⁶⁴Zn) would equal 51.37% minus(10%×51.37), which equals 46.233%, and the ⁶⁴Zn atom percent in thesample would be (100%−46.233%), which equals 53.767%. The sample wouldthus be characterized as enriched for ⁶⁴Zn by 10% of D.

The authors of the present invention have discovered that a compositionthat comprises at least one light isotope selected from the groupconsisting of ¹H, ¹²C, ¹⁶O, ¹⁴N, ³⁹K, ²⁴Mg, ⁶⁴Zn, ⁸⁵Rb, ²⁸Si, ⁵⁴Fe,⁹²Mo, ⁷⁴Se, ⁵⁸Ni, ⁷⁰Ge, ⁵²Cr, ⁶³Cu, and ⁴⁹V, wherein the composition isenriched for the at least one light isotope relative to the naturalabundance of the isotope, has pronounced bacteriostatic and bactericidaleffects approximately equal to or greater than those provided byconventional antibiotics. Thus, the antibacterial compositions of thepresent invention inhibit the growth of and/or kill bacteria. Inaddition to being enriched for a light isotope as described above, thecomposition may comprise one or more additional active ingredients, aswell as water and inert auxiliary ingredients such as carriers, diluentsand the like which are used to formulate the said composition and may bepharmaceutically acceptable or pharmaceutically unacceptable (which areused as intermediates in the preparation of pharmaceutically acceptableagents). The ability of a chemical element enriched for a light isotopeto exhibit antibacterial activity was revealed by the authors of thepresent invention and has not been described previously in theliterature.

As used herein, the terms “treat,” “treating,” “treatment of” acondition encompass performing an act (such as administering thecomposition of the invention) in order to cure, eradicate, or diminishthe severity of, the condition treated. These terms thus encompassaccomplishing any one or more of curing, eradicating, and diminishingthe severity of the condition treated. As used herein, the terms“prevent,” “preventing,” “prevention of” a condition encompassperforming an act (such as administering the composition of theinvention) in order to prevent the occurrence of the condition anddiminish the severity of the condition if it occurs subsequent to theact. These terms thus encompass accomplishing any one or more of whollypreventing the condition from occurring and diminishing the severity ofthe condition if it occurs subsequent to the act.

For reference with respect to the invention, the above-listed isotopesare considered to have the natural abundances, on a mole-fraction basis,shown in the following table. The table also shows the correspondingpercentages preferred for use in the compositions of the invention, on amole-fraction basis (lower limits are provided; in every case, themaximum theoretical upper limit is 100%). For example, in a compositionof the invention that uses a therapeutic amount of ⁶⁴Zn, the zinc in thecomposition preferably would contain at least about 90% ⁶⁴Zn.Compositions that contain isotopes with lower levels of enrichment mayalso be effective and are within the scope of the invention.

Isotope Natural abundance (%) % for therapeutic use ¹H 99.9885 at least99.99% ¹²C 98.93 at least 99.9% ¹⁴N 99.632 at least 99.9% ¹⁶O 99.757 atleast 99.9% ²⁴Mg 78.99 at least about 95%* ²⁸Si 92.2297 at least about95% ³⁹K 93.2581 at least about 98% ⁵⁰V 0.250 at least about 35% ⁵²Cr83.789 at least about 90% ⁵⁴Fe 5.845 at least about 80%* ⁵⁸Ni 68.0769 atleast about 90%* ⁶³Cu 69.17 at least about 90%* ⁶⁴Zn 48.63 at leastabout 90%* ⁷⁰Ge 20.84 at least about 80%* ⁷⁴Se 0.89 at least about 50%*⁸⁵Rb 72.17 at least about 90%* ⁹²Mo 14.84 at least about 80%* *In someembodiments, an enrichment level about 10 percentage points lower may beused for this isotope for therapeutic application and preferably forprophylactic use. For example, for ⁶⁴Zn, a composition in which the zinccontains at least about 80% ⁶⁴Zn may be administered for therapeutic orprophylactic purposes such as preventing bacterial infection of a woundor cut.

The term “minimum inhibitory concentration,” or “MIC,” as used herein,represents the lowest concentration of a test drug that prevents growthof a test culture. The minimum inhibitory concentration of abacteriostatic or bactericidal agent is that which causes completesuppression of visible growth of a given microorganism in media understandard test conditions. It is expressed in micrograms (“mcg”)/ml or inunits of activity.

The term “minimum bactericidal concentration,” or “MBC,” as used herein,represents the lowest concentration of a test drug that causes abactericidal effect, i.e. the concentration that results in the death ofa bacterium test strain, such as a standard bacterium test strain. Itmay be expressed, for example, in mg/l or mcg/ml.

In an antibacterial composition of the invention, the composition isenriched for at least one light isotope selected from the group thatincludes ¹H, ¹²C, ₁₆O, ¹⁴N, ³⁹K, ²⁴Mg, ⁶⁴Zn, ⁸⁵Rb, ²⁸Si, ⁵⁴Fe, ⁹²Mo,⁷⁴Se, ⁵⁸Ni, ⁷⁰Ge, ⁵²Cr, ⁶³Cu, ⁵⁰V or any combination thereof. At leastone light isotope may be present as a component of a chemical compound,such as the salt of an organic or inorganic acid, which ispharmaceutically acceptable and can be administered to humans andveterinary animals (such as veterinary mammals). Exemplary salts includethe chloride, citrate, sulfate, aspartate, glutamate, asparaginate andethylene diamine disuccinic acid (referred to herein interchangeably as“EDDS” and “EDDA”) salts of the light isotope, and hydrates of suchsalts. For example, zinc enriched for ⁶⁴Zn may be present in the form ofthe salt zinc aspartate or the salt zinc asparaginate.

The antibacterial composition of the invention may be prepared by makinga compound that is enriched for a light isotope, such as the salt of anorganic or inorganic acid and the light isotope, purifying the obtainedcompound by standard methods, and subsequent preparation of the claimedantibacterial composition in any appropriate form, such as an aqueoussolution. Such methods are well known and the person of ordinary skillin the art can prepare a compound containing a light isotope of aparticular chemical element, its salt in particular. The preparationprocess of the complex of aspartic acid and zinc which is enriched forthe isotope ⁶⁴Zn is described in the Examples below. The lightisotope-enriched compound may be administered as a component oringredient of any convenient dosage form. Such dosage forms includetopical dosage forms such as solutions, sprays, lotions, salves,ointments, gels, creams, soaps, shampoos, and foams, oral dosage formssuch as tablets, capsules, syrups, suspensions, lozenges, gums, sprays,patches, and solutions, and conventional dosage forms suitable for otherconventional routes of administration. Conventional dosage forms arewell-known to the person of ordinary skill in the art. Examples of suchdosage forms and their preparation are described in, for example, LoydV. Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and DrugDelivery Systems (8th ed. 2005) (Lippincott Williams & Wilkins), andpublications cited therein.

The antibacterial composition of the invention may contain water assolvent. The antibacterial composition of the invention may be in theform of an aqueous solution to be administered by any suitable route,such as orally and topically, or in the form of a gel, salve, ointment,paste, cream, foam, lotion, drops, or other topical composition. Thecomposition may further include any suitable excipient known to theperson of ordinary skill in the art, including solvents, binders,lubricants, emulsifiers, detergents, surfactants, buffers, stabilizers,and preservatives. These are described in commonly used references, suchas the Handbook of Pharmaceutical Excipients.

The concentration of the light isotope-enriched element in a compositionof the invention, relative to the total weight of the composition,varies according to conventional composition weights and the dosage ofthe light isotope-enriched element. Appropriate dosages of the lightisotope-enriched element are set forth below. Preferably the compositionof the invention comprises an effective amount of at least one lightisotope, wherein “effective amount” refers to that amount that eithersuppresses, partially or completely, the growth of bacteria at theaffected site, or kills some or all of the bacteria at the affectedsite. As stated above, the quantity of light isotope that is effectiveis proportional to the quantity of the corresponding element that ispresent in the body. Where the body contains a relatively large quantityof the element, a correspondingly relatively large amount of theelement's light isotope will be required to provide an effective dosageamount. On the other hand, where the body contains a relatively smallquantity of the element, a correspondingly relatively small amount ofthe element's light isotope will be required to provide an effectivedosage amount. These quantities are reflected in the “guidance amounts”for each element, the recommended amount for daily human consumption, asdetailed below.

In certain embodiments, the preferred dosage of any of the lightisotopes is proportional to various authoritative daily ingestionguidances (e.g. recommended dietary allowance (USRDA), adequate intake(AI), recommended dietary intake (RDI)) of the corresponding element.The light isotope dosage is preferably between about ½ and about 20times the guidance amount of the corresponding element, more preferablybetween about 1 and about 10 times the guidance amount, even morepreferably between about 1 and about 3 times the guidance amount. Thus,in preferred embodiments, a single dose of a composition of theinvention for daily administration would be formulated to comprise aquantity within these ranges, such as about ½, about 1, about 3, about5, about 10, and about 20 times the guidance amount. These amountsgenerally are for oral intake or topical application. In someembodiments, the preferred intravenous dosage is lower, such as fromabout 1/10 to about ½ the guidance amount. In another embodiment,intravenous treatment of sepsis preferably employs a zinc dosage of fromabout 10 to about 100 times the daily guidance amount. Doses at the lowend of these ranges are appropriate for anyone with a heightenedsensitivity to a specific element or class of elements (e.g., those withkidney problems). For zinc, the guidance amount ranges from 2 mg ininfants to 8-11 mg (depending on sex) for ages 9 and up. Guidanceamounts for some of the elements used in the compositions of theinvention are presented below based on information obtained fromhttps://ods.od.nih.gov/factsheets/list-all/ andhttps://health.gov/dietaryguidelines/2015/guidelines/appendix-7/,summarized below. Daily dosages discussed throughout this applicationmay be subdivided into fractional dosages and the fractional dosagesadministered the appropriate number of times per day to provide thetotal daily dosage amount (e.g. ½ the daily dose administered twicedaily, ⅓ the daily dose administered three times daily, etc.).

Element/Isotope guidance amount, daily magnesium/ 30-420 mg ²⁴Mg(400-420 mg in males 14+; 310-360 mg in females 14+) potassium/ 1 to 3years: 3 g/day ³⁹K 4 to 8 years: 3.8 g/day 9 to 13 years: 4.5 g/day 14to 18 years: 4.7 g/day Age 19 and older: 4.7 g/day chromium/ Hexavalentchromium should be avoided. ⁵²Cr Chromium complexes are preferred fororal administration (e.g. picolinate, dinicocysteinate, as nicotinicacid complex). For parenteral administration, chromic chloride at 4mcg/ml may be used. 0-6 mos. 0.2 mcg 7-12 mos. 5.5 mcg 1-3 yrs 11 mcg4-8 yrs 15 mcg 9-13 yrs females: 21 mcg, males: 25 mcg 14-18 yrsfemales: 24 mcg, males; 35 mcg 19-50 yrs females: 25 mcg, males: 35mcg >50 yrs females: 20 mcg, males: 30 mcg Iron/ Birth to 6 months 0.27mg ⁵⁴Fe 7-12 months 11 mg 1-3 years 7 mg 4-8 years 10 mg 9-13 years 8 mg14-18 years males: 11 mg, females: 15 mg 19-50 years males: 8 mg,females: 18 mg Adults 51 years and older 8 mg Copper/ adequate: ⁶³Cu 0to 6 months: 200 mcg 7 to 12 months: 220 mcg recommended: 1 to 3 years:340 mcg 4 to 8 years: 440 mcg 9 to 13 years: 700 mcg 14 to 18 years: 890mcg 19 and older: 900 mcg Zinc/ Birth to 6 months 2 mg ⁶⁴Zn 7 months-3years 3 mg Children 4-8 years 5 mg Children 9-13 years 8 mg 14-18 years(boys) 11 mg 14-18 years (girls) 9 mg Adults (men) 11 mg Adults (women)8 mg Selenium/ Birth to 6 months 15 mcg ⁷⁴Se 7 months-3 years 20 mcgChildren 4-8 years 30 mcg Children 9-13 years 40 mcg 14 years and older55 mcg

For purposes of the invention, for the following substances, thefollowing amounts are considered to be benchmark daily intakes:rubidium: between about 1 and 2 mg per day; “light water” (waterenriched for ¹H and thus depleted for deuterium and/or tritium): about400 micrograms; silicon: about 10 mg; molybdenum: about 1.5 mg;germanium: about 1 mg; nickel: about 100 mcg; vanadium: about 40 mcg.Thus, a composition of the invention that contains light rubidium orlight water, for example, preferably contains ⁸⁵Rb_(e) or light water(¹H_(e2)O), respectively, in an amount between about 1 times and about20 times these amounts, more preferably between about 1 and about 10times these amounts, and even more preferably between about 1 and about3 times these amounts.

Based on the above, in certain embodiments, a composition of theinvention containing ⁶⁴Zn_(e) as the active ingredient, prepared foradministration to a male 19 years of age or older, preferably contains,in a single dose, between about 11 mg and about 220 mg ⁶⁴Zn_(e) (zincenriched for ⁶⁴Zn), more preferably between about 11 mg and about 110 mg⁶⁴Zn_(e), even more preferably between about 11 mg and about 33 mg⁶⁴Zn_(e). Such a composition may be, for example, for oraladministration, such as a tablet or capsule, or for topicaladministration, such as a cream, gel, ointment, or lotion (optionallycontaining DMSO or other absorption-enhancing agent and otherappropriate excipients).

In certain preferred embodiments, the daily dosages of ⁶⁴Zn_(e) in acomposition of the invention, such as a tablet, capsule, salve, cream,lotion, or ointment, comprise between about 10 and about 50 mg of⁶⁴Zn_(e), such as about 15 mg, about 30 mg, or about 45 mg of ⁶⁴Zn_(e),which may be elemental or in the form of ⁶⁴Zn_(e) asparaginate, ⁶⁴Zn_(e)aspartate, or another pharmaceutically acceptable ⁶⁴Zn_(e) salt orcomplex. Such compositions preferably contain, in addition to the⁶⁴Zn_(e) compound, excipients suitable to the formulation type. Inanalogous preferred embodiments, the daily dosages of another lightisotope may be determined relative to these dosages and the relativeguidance amounts of ⁶⁴Zn_(e) and the other light isotope. For example,if the guidance amount of another light isotope were one-half (½) thatof zinc, then preferred daily doses of the other light isotope in acomposition of the invention would be between about 5 mg and about 25mg, such as about 7.5 mg, about 15 mg, or about 22.5 mg, in elementalform or as a pharmaceutically acceptable salt or complex.

In some embodiments, a composition of the invention may contain two ormore compounds that are each enriched for a light isotope. Thepercentages and masses above may represent each of the lightisotope-enriched compounds and may alternatively represent their totalpercentage or mass.

The composition of the invention may include an additional antibacterialagent, as well as auxiliary agents which improve the stability andantibacterial properties of the composition and are generally present inmany finished pharmaceutical products.

Compositions that contain zinc are known and include topicalformulations that contain 20% or 40% w/w zinc oxide and oralformulations such as tablets and capsules that contain 30 mg or 50 mgzinc in various forms. In an embodiment, the present invention providescomparable compositions in which the zinc is enriched for ⁶⁴Zn. Forexample, the zinc in such compositions may contain at least about 90%⁶⁴Zn, such as between about 90% and about 99.9% ⁶⁴Zn, such as about 90%,about 95%, about 99%, or about 99.9% ⁶⁴Zn, on a mole fraction basis.Examples of such compositions include: a paste that contains betweenabout 20% w/w and about 40% w/w ⁶⁴Zn_(e) oxide, such as about 20%, about30%, or about 40% w/w ⁶⁴Zn_(e) oxide; an ointment that contains about20% w/w ⁶⁴Zn_(e) oxide; tablets and capsules that contain between about30 mg and about 50 mg of ⁶⁴Zn_(e), such as about 30, about 40, or about50 mg ⁶⁴Zn_(e), present in the form of zinc gluconate, zinc bisglycinatechelate, or any pharmaceutically acceptable zinc salt such as thoseenumerated above (aspartate, asparaginate, glutamate, EDDA, etc.). Suchcompositions preferably also contain excipients suitable to eachformulation type. Examples of such excipients and representative paste,ointment, tablet and capsule compositions, and their preparation, aredisclosed, for example, in Ansel's Pharmaceutical Dosage Forms and DrugDelivery Systems (8th ed. 2005) (Lippincott Williams & Wilkins)(capsules and tablets are discussed, for example, at pages 204-75, andointments and pastes are discussed, for example, at pages 276-97; bothsections are incorporated by reference herein in their entirety), andpublications cited therein. Dosage amounts of any topical compositionsof the invention preferably vary with skin thickness at the site ofadministration, with higher dosage amounts being used on thicker skinand lower dosages on thinner skin.

The pH of the said composition, when aqueous (including emulsions suchas oil-in-water and water-in-oil emulsions), may be between about 2 andabout 10, such as between about 2 and about 4, between about 4 and about6, between about 6 and about 8, or between about 8 and about 10. Forexample, the composition may have a pH of about 2, about 3, about 4,about 5, about 6, about 7, or about 8, as appropriate for the route ofadministration and site of administration.

The antibacterial composition enriched for a light isotope as describedherein slows, reduces, or stops the growth of bacteria at the targetedsite or kills such bacteria, prevents bacterial infection, and/oreliminates bacterial infection wherein the said infection is caused byat least one bacterium. The bacteria or bacterium may be eithergram-negative or gram-positive. Examples of infection-causing genera andspecies that the composition of the invention is effective againstinclude Bacillus, Escherichia coli, Acinetobacter, Salmonella,Haemophilus influenzae, Vibrio parahaemolyticus, Enterococcus,Pneumococcus, Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Group AStreptococcus, Group B Streptococcus, Group C/G Streptococcus, Listeriamonocytogenes, Klebsiella pneumoniae, Shigella, Vibrio cholerae,Pseudomonas aeruginosa.

The invention thus provides a method of treating a condition that has abacterial component, including those that are caused by or characterizedby a bacterial infection or excessive bacterial growth, wherein themethod comprises administering a composition of the invention to aperson or veterinary animal, such as a veterinary mammal, that exhibitssuch a condition. In an embodiment of this method of the invention, thebacterial component of the condition comprises one or more gram-positiveor gram-negative bacterium, including the bacteria enumerated in thepreceding paragraph. In other embodiments of this method of theinvention, the condition treated is acne, such as acne vulgaris, orsepsis. The invention also provides a method of preventing a conditionthat has a bacterial component, including those that are caused by orcharacterized by a bacterial infection or excessive bacterial growth,wherein the method comprises administering a composition of theinvention to a person or veterinary animal, such as a veterinary mammal,who is in need of such treatment. In an embodiment, the method comprisesadministering the composition of the invention to a site that is likelyto become the site of a bacterial infection or excessive bacterialgrowth, such as an open wound such as a cut, or the site of a surgicalincision, or to an area of skin in the vicinity of acne inflammation orwhere acne inflammation has previously occurred, or to another areaprone to infection, in order to kill bacteria and/or prevent bacterialgrowth at the site of administration. In various embodiments, suchadministration can be oral, topical or by injection. The compositions ofthe invention may also be used to treat sepsis, preferably viaintravenous administration. In the context of the invention, “excessivebacterial growth” means greater growth than would ordinarily be expectedat the site that is intended to be treated by administering thecomposition. In the context of the invention, “veterinary animal” refersto an animal that a veterinarian would treat, including, but not limitedto, pets such as dogs and cats, farm animals such as cows and horses,and zoo animals such as monkeys, chimpanzees, and orangutans, lions,tigers, and elephants.

Thus, the invention provides a method of treating or preventing acondition that has a bacterial component, including those that arecaused by or characterized by a bacterial infection or excessivebacterial growth comprising administering an effective amount of thecomposition to a human patient or veterinary animal, such as aveterinary mammal, in need of such treatment. The composition may beadministered topically, orally, or by injection, and is formulatedaccordingly. The invention further provides compositions for use in thetreatment or prevention of a condition that has a bacterial component,including those that are caused by or characterized by a bacterialinfection or excessive bacterial growth, wherein the composition is foradministration to a human patient or veterinary animal, such as aveterinary mammal, in need of such treatment. Such conditions includethose detailed in the preceding paragraph. The composition may beadministered topically, orally, or by injection, and is formulatedaccordingly.

An advantage of the proposed composition is that the antibacterialcomposition which comprises and is enriched for at least one lightisotope selected from the group consisting of ¹H, ¹²C, ¹⁶O, ¹⁴N, ³⁹K,²⁴Mg, ⁶⁴Zn, ⁸⁵Rb, ²⁸Si, ⁵⁴Fe, ⁹²Mo, ⁷⁴Se, ⁵⁸Ni, ⁷⁰Ge, ⁵²Cr, ⁶³Cu, ⁵⁰Vand any combination thereof, does not cause any toxic effects whenadministered, unlike many other antibacterial agents. These elementsplay an important physiological role in many organisms, includinghumans. Consequently, the composition of the invention provides, inaddition to its bactericidal and bacteriostatic activity, a number ofadditional advantages associated with the optimization of the variousbiological functions that make use of these elements, includingcatalytic, structural and regulatory functions.

The present invention will further be more fully disclosed by referenceto the following Examples. The Examples are given as illustrations andshould not be construed to limit the scope of the invention in any way.The following Examples present data relating to ⁶⁴Zn, the light isotopeof zinc.

EXAMPLES Example 1 Preparation Process of ⁶⁴Zn_(e) Aspartate

⁶⁴Zn_(e) aspartate (racemic) having the following formula (in which“⁶⁴Zn²⁺” refers, in this instance, to Zn²⁺ enriched for ⁶⁴Zn) wasprepared in the experiment.

At the first stage, zinc oxide enriched for ⁶⁴Zn was prepared using⁶⁴Zn_(e) sulfate as the starting compound.

⁶⁴Zn_(e)SO₄+2NaHCO₃→⁶⁴Zn_(e)O+Na₂SO₄+2CO₂+H₂O

For this purpose, ⁶⁴Zn_(e) sulfate (zinc was at least 99.9% ⁶⁴Zn,although ⁶⁴Zn_(e) of lower purity may be effective) in an amount of 0.01mole) was dissolved in 150 ml of water (T=50-70° C.) wherein 1.68 g(0.02 mole) of sodium bicarbonate was added in small portions, toprevent severe foaming, with constant stirring in a magnetic stirrer.After completion of foaming the solution was stirred for another 30minutes and then left for 1 hour until a white precipitate was formed.During this process, the temperature was maintained at about 60° C. toprevent crystallization of sodium sulfate. The solution with theprecipitate, which precipitate was ⁶⁴Zn_(e)O, was then filtered withoutcooling. The resulting precipitate—⁶⁴Zn_(e)O—was washed with warmdemineralized water (T=40-50° C.) and dried to constant weight in adesiccator over the dehydrating agent phosphorus pentoxide.

After that, 425 ml of demineralized water was poured into a 1 literflask and heated under reflux to 80° C. 1.33 g (0.01 mole) of asparticacid was dissolved in water with stirring by a magnetic stirrer. Afteraspartic acid was completely dissolved, 0.8 g (0.01 mole) of ⁶⁴Zn_(e)Oobtained at the previous stage was added to the clear solution. Themixture was stirred with heating to 80° C. for 1½-2 hours till completedissolution of ⁶⁴Zn_(e)O. The reaction formula is shown below:

If the precipitate (⁶⁴Zn_(e) oxide) was not dissolved completely, thesolution was filtered and the undissolved ⁶⁴Zn_(e)O was collected anddried to its constant weight to determine the ⁶⁴Zn_(e) complexconcentration in the resulting solution. The solution was transferredinto a volumetric measure and made up to a volume of 425 ml usingdemineralized water. 425 ml of ⁶⁴Zn_(e)-aspartic acid complex containing⁶⁴Zn_(e) in the amount of approximately 0.0015 g ⁶⁴Zn_(e) (1.5 mg⁶⁴Zn_(e))/ml was thus prepared.

In Examples 2 through 4 that follow, in the ⁶⁴Zn_(e) compounds that wereused in the experiments described, the zinc was about 99.4% ⁶⁴Zn on amole fraction basis.

Example 2 Determination of MIC and MBC of the Antibacterial CompositionBased on Various ⁶⁴Zn_(e) Compounds

The minimum inhibitory concentration (MIC) and minimum bactericidalconcentration (MBC) of the antibacterial composition enriched for thelight isotope zinc ⁶⁴Zn_(e) was evaluated in the experiment. Fivesamples of ⁶⁴Zn_(e) (⁶⁴Zn_(e) citrate, ⁶⁴Zn_(e) salt with EDDA, ⁶⁴Zn_(e)sulfate, ⁶⁴Zn_(e) asparaginate, ⁶⁴Zn_(e) glutamate) were tested. S.aureus ATCC 25923, E. coli ATCC 25922 and P. aeruginosa ATCC 27853 wereused as test cultures.

The studies were carried out using the method of serial dilutions inbroth. To determine sensitivity of microorganisms to the antibacterialcomposition, 0.5 ml of broth was placed into each tube. The number oftubes was determined based on the desired number of dilutions and wasincreased by one for the “negative” control (no test composition).Series of two-fold serial dilutions of the test variants of theantibacterial composition were used and concentrations ranging from 450mcg ⁶⁴Zn_(e)/ml to 0.000107 mcg ⁶⁴Zn_(e)/ml were obtained.

Reference test strains of S. aureus ATCC 25923, E. coli ATCC 25922 andP. aeruginosa ATCC 27853 were incubated in Mueller-Hinton liquid brothat 37° C. for 24 hours. Standard bacterial suspension equivalent to 0.5McFarland standard diluted 100-fold with the broth was used for theinoculation of the tubes after which the concentration of microorganismin it was approximately 10⁶ CFU/ml.

0.5 ml of inoculum was added to each tube containing 0.5 ml of theappropriate dilution of a solution of the ⁶⁴Zn-enriched salt ofinterest. 0.5 ml of inoculum also was added to one tube with 0.5 ml ofbroth that did not contain the ⁶⁴Zn-enriched salt of interest (the“negative” control). The final concentration of the microorganism ineach tube was approximately 5×10⁵ CFU/ml.

The tubes were incubated at 35° C. in air for 16-20 or 20-24 hours(depending on the bacterial strain). The tube of the negative controlwas placed in a refrigerator at +4° C. where it was stored till theanalysis of the results.

To determine the presence of microorganism growth the test tubes withinoculum were viewed under transmitted light. Transmitted light wasmeasured by spectrophotometry. The growth of culture in the presence ofantibacterial compositions based on ⁶⁴Zn_(e) was compared with thereference test tube (“negative” control), containing the originalinoculum and stored in the refrigerator. The MIC was determined as thelowest concentration of ⁶⁴Zn_(e) that inhibits visible growth of themicroorganism. Both gram-positive (S. aureus) and gram-negative bacteria(P. aeruginosa, E. coli) were used in the experiments described below.

The experimental results are shown in Tables 1-5. The tables indicatethe presence or absence of visible growth as a function of test cultureand degree of dilution of the concentration of the antibacterialsubstance (zinc salt enriched for ⁶⁴Zn). The salts used were citrate,EDDA, sulfate, aspartate, and glutamate.

TABLE 1 Antibacterial activity of the composition on the basis of⁶⁴Zn_(e) citrate Initial concentration of the sample: 900 mcg⁶⁴Zn_(e)/ml Dilution No. 1 2 3 4 5 6 7 8 log₂(Dilution factor)^(†) 1 2 34 5 6 7 8 Concentration of ⁶⁴Zn_(e), mcg/ml* Test cultures 450 225 112.556.25 28.13 14.06 7.03 3.52 S. aureus −** − − − − − − − E. coli − − − −− − − − P. aeruginosa − − − − − − + + Dilution No. 9 10 11 12 13 14 1516 log₂(Dilution factor) 9 10 11 12 13 14 15 16 Concentration of⁶⁴Zn_(e), mcg/ml Test cultures 1.76 0.88 0.44 0.22 0.11 0.055 0.0270.014 S. aureus − − − − − − − + E. coli − − − − − − − + P.aeruginosa + + + + + + + + Dilution No. 17 18 19 20 21 22 23log₂(Dilution factor) 17 18 19 20 21 22 23 Concentration of ⁶⁴Zn_(e),mcg/ml Test cultures 0.0069 0.0034 0.0017 0.00086 0.00043 0.000220.00011 MIC, mcg/ml S. aureus + + + + + + + 0.0275 E. coli + + + + + + +0.0275 P. aeruginosa + + + + + + + 14.062 ^(†)Dilution factor = 2^(n),where n is the number in the table cell. For example, where 5 is thenumber in the cell, the dilution factor is 2⁵ = 32: the concentration ofthe substance is 1/32 of the original or initial concentration.*Non-integer substance concentrations are rounded off. **“−” indicatesno visible growth; “+” indicates presence of visible growth.

TABLE 2 Antibacterial activity of the composition on the basis of⁶⁴Zn_(e) EDDS salt (⁶⁴Zn_(e) salt of ethylenediamine-N,N′-disuccinicacid, chemical name 2-[2-[[(1)-1-carboxy-2-carboxylatoethyl]amino]ethylamino]butanedioate) Initial concentration ofthe sample: 3000 mcg ⁶⁴Zn_(e)/ml Dilution No. 1 2 3 4 5 6 7 8log₂(Dilution factor)^(†) 1 2 3 4 5 6 7 8 Concentration of ⁶⁴Zn_(e),mcg/ml* Test cultures 1500 750 375 187.5 93.75 46.88 23.44 11.72 S.aureus −** − − − − − − − E. coli − − − − − − − − P. aeruginosa − − − − −− − − Dilution No. 9 10 11 12 13 14 15 16 log₂(Dilution factor) 9 10 1112 13 14 15 16 Concentration of ⁶⁴Zn_(e), mcg/ml Test cultures 5.86 2.931.46 0.73 0.37 0.18 0.092 0.046 S. aureus − − − − − ± ± + E. coli − − −− − − − − P. aeruginosa − − − + + + + + Dilution No. 17 18 19 20 21 2223 log₂(Dilution factor) 17 18 19 20 21 22 23 Concentration of ⁶⁴Zn_(e),mcg/ml Test cultures 0.023 0.011 0.0057 0.0029 0.0014 0.00072 0.00036MIC, mcg/ml S. aureus + + + + + + + 0.366 E. coli − + + + + + + 0.0229P. aeruginosa + + + + + + + 1.465 ^(†)Dilution factor = 2^(n), where nis the number in the table cell. For example, where 5 is the number inthe cell, the dilution factor is 2⁵ = 32: the concentration of thesubstance is 1/32 of the original concentration. *Non-integer substanceconcentrations are rounded off. **“−” indicates no visible growth; “+”indicates presence of visible growth.

TABLE 3 Antibacterial activity of the composition on the basis of⁶⁴Zn_(e) sulfate Initial concentration of the sample: 3000 mcg ⁶⁴Zn_(e)/ml Dilution No. 1 2 3 4 5 6 7 8 log₂(Dilution factor)^(†) 1 2 3 45 6 7 8 Concentration of ⁶⁴Zn_(e), mcg/ml* Test cultures 1500 750 375187.5 93.75 46.88 23.44 11.72 S. aureus −** − − − − − − − E. coli − − −− − − − − P. aeruginosa − − − − − − + + Dilution No. 9 10 11 12 13 14 1516 log₂(Dilution factor) 9 10 11 12 13 14 15 16 Concentration of⁶⁴Zn_(e), mcg/ml Test cultures 5.86 2.93 1.46 0.73 0.37 0.18 0.092 0.046S. aureus − − − − − ± ± + E. coli − − − − − − − + P.aeruginosa + + + + + + + + Dilution No. 17 18 19 20 21 22 23log₂(Dilution factor) 17 18 19 20 21 22 23 Concentration of ⁶⁴Zn_(e),mcg/ml Test cultures 0.023 0.011 0.0057 0.0029 0.0014 0.00072 0.00036MIC, mcg/ml S. aureus + + + + + + + 0.366 E. coli + + + + + + + 0.0916P. aeruginosa + + + + + + + 46.875 ^(†)Dilution factor = 2^(n), where nis the number in the table cell. For example, where 5 is the number inthe cell, the dilution factor is 2⁵ = 32: the concentration of thesubstance is 1/32 of the original concentration. *Non-integer substanceconcentrations are rounded off. **“−” indicates no visible growth; “+”indicates presence of visible growth.

TABLE 4 Antibacterial activity of the composition on the basis of ⁶⁴Zn_(e) aspartate Initial concentration of the sample: 1500 mcg/mlDilution No. 1 2 3 4 5 6 7 8 log₂(Dilution factor)^(†) 1 2 3 4 5 6 7 8Concentration of ⁶⁴Zn_(e), mcg/ml* Test cultures 750 375 187.5 93.7546.88 23.44 11.72 5.86 S. aureus −** − − − − − − − E. coli − − − − − − −− P. aeruginosa − − − − − − − − Dilution No. 9 10 11 12 13 14 15 16log₂(Dilution factor) 9 10 11 12 13 14 15 16 Concentration of ⁶⁴Zn_(e),mcg/ml Test cultures 2.93 1.46 0.73 0.37 0.18 0.092 0.046 0.023 S.aureus − − − − − − − − E. coli − − − − − − − − P. aeruginosa − −− + + + + + Dilution No. 17 18 19 20 21 22 23 log₂(Dilution factor) 1718 19 20 21 22 23 Concentration of ⁶⁴Zn_(e), mcg/ml Test cultures 0.0110.0057 0.0029 0.0014 0.00072 0.00036 0.00018 MIC, mcg/ml S. aureus −− + + + + + 0.0057 E. coli + + + + + + + 0.0229 P.aeruginosa + + + + + + + 0.732 ^(†)Dilution factor = 2^(n), where n isthe number in the table cell. For example, where 5 is the number in thecell, the dilution factor is 2⁵ = 32: the concentration of the substanceis 1/32 of the original concentration. *Non-integer substanceconcentrations are rounded off. **“−” indicates no visible growth; “+”indicates presence of visible growth.

TABLE 5 Antibacterial activity of the composition on the basis of⁶⁴Zn_(e) glutamate Initial concentration of the sample: 1500 mcg/mlDilution No. 1 2 3 4 5 6 7 8 log₂(Dilution factor)^(†) 1 2 3 4 5 6 7 8Concentration of ⁶⁴Zn_(e), mcg/ml* Test cultures 750 375 187.5 93.7546.88 23.44 11.72 5.86 S. aureus −** − − − − − − − E. coli − − − − − − −− P. aeruginosa − − − − − − − − Dilution No. 9 10 11 12 13 14 15 16log₂(Dilution factor) 9 10 11 12 13 14 15 16 Concentration of ⁶⁴Zn_(e),mcg/ml Test cultures 2.93 1.46 0.73 0.37 0.18 0.092 0.046 0.023 S.aureus − − − − − − − + E. coli − − − − − − − − P. aeruginosa − − − −− + + + Dilution No. 17 18 19 20 21 22 23 log₂(Dilution factor) 17 18 1920 21 22 23 Concentration of ⁶⁴Zn_(e), mcg/ml Test cultures 0.011 0.00570.0029 0.0014 0.00072 0.00036 0.00018 MIC, mcg/ml S.aureus + + + + + + + 0.0458 E. coli − − + + + + + 0.0057 P.aeruginosa + + + + + + + 0.183 ^(†)Dilution factor = 2^(n), where n isthe number in the table cell. For example, where 5 is the number in thecell, the dilution factor is 2⁵ = 32: the concentration of the substanceis 1/32 of the original concentration. *Non-integer substanceconcentrations are rounded off. **“−” indicates no visible growth; “+”indicates presence of visible growth.

To evaluate the bactericidal action of the antibacterial compositionbased on light isotope ⁶⁴Zn_(e) the minimum bactericidal concentration(MBC) was determined by plating out 0.1 ml of the contents of each tubecontaining the antibacterial composition on Mueller-Hinton medium. Theincubation was at 37° C. for 24 h.

The MBC of the test samples was determined as the lowest concentrationat which there was no growth on Mueller-Hinton agar medium. Theestablished MBC values are given in Table 6.

TABLE 6 The MBC for all samples of the antibacterial composition basedon light isotope ⁶⁴Zn_(e) for test cultures MBC, mcg ⁶⁴Zn_(e)/ml⁶⁴Zn_(e) ⁶⁴Zn_(e) ⁶⁴Zn_(e) ⁶⁴Zn_(e) ⁶⁴Zn_(e) Test culture citrate EDDSsulfate asparaginate glutamate S. aureus 0.028* 0.73* 0.37* 0.023*0.046* E. coli 0.44* 2.93* 1.46* 0.73* 0.37* P. aeruginosa 112.5 375375 >750 >750 *Value is rounded off.

The data presented above show that the antibacterial composition of theinvention had a pronounced bacteriostatic (Tables 1-5) and bactericidalactivity (Table 6). All samples of the composition demonstrated goodindicators of bacteriostatic activity against S. aureus and E. coli. Asfor P. aeruginosa, the best results were recorded for the antibacterialcomposition which included ⁶⁴Zn_(e) in the form of salt with EDDA,aspartic acid and glutamic acid (see FIG. 1). Bactericidal activity ofthe antibacterial composition against S. aureus and E. coli wassufficiently high in all the samples. The samples of the antibacterialcomposition based on ⁶⁴Zn_(e) in the form of citrate, sulfate and saltwith EDDA (see FIG. 2) showed satisfactory bactericidal activity againstP. aeruginosa.

Example 3 Comparison of Properties of the Antibacterial Composition witha Zinc-Based Composition with Natural Distribution of Isotopes

For further studies of the antibacterial properties of a composition inaccordance with the invention to confirm the fact that itsbacteriostatic and bactericidal activities are due to the enrichment ofthe light isotope ⁶⁴Zn in the composition, a parallel experiment wascarried out to compare the antibacterial properties of a compositionthat includes zinc that is not enriched for ⁶⁴Zn. That is, ⁶⁴Zn_(e) inthe form of a salt was compared with a composition that contains zincwith a natural distribution of isotopes. The conditions of theexperiment were the same as those described in Example 1. Theantibacterial composition of the invention containing ⁶⁴Zn_(e) in theform of a salt with EDDA and in the form of ⁶⁴Zn_(e) sulfate served asthe samples for the comparison. Relevant samples containing salt ofnatural zinc with EDDA and natural zinc sulfate were used for thecomparison. The findings of the study of the bacteriostatic activity(MIC values) are shown in Table 7 and FIG. 3; the findings of the studyof the bactericidal activity (MBC values) are shown in Table 8 and FIG.4.

TABLE 7 Comparative figures of MIC of samples containing ⁶⁴Zn_(e) andsamples with natural zinc MIC, zinc/ml ⁶⁴Zn_(e) Natural zinc - ⁶⁴Zn_(e)Natural zinc - Test culture EDDS EDDS sulfate sulfate S. aureus 0.7393.75 0.366 93.75 E. coli 0.18 375 0.0916 187.5 P. aeruginosa 5.86 37546.875 750

TABLE 8 Comparative figures of MBC of samples containing ⁶⁴Zn_(e) andsamples with natural zinc MBC, mcg zinc/ml ⁶⁴Zn_(e) Natural zinc as⁶⁴Zn_(e) Natural zinc Test culture EDDS part of EDDS sulfate sulfate S.aureus 0.7324 375 0.3662 >750 E. coli 2.9296 >750 1.4648 >750 P.aeruginosa 375 >750 375 >750

The data above show that the antibacterial composition of the inventioncontaining ⁶⁴Zn-enriched zinc demonstrated a bacteriostatic activitywhich is significantly higher than that of the zinc-based preparationswith the natural isotope distribution (by a factor of about 15 to about2000 maximum). The bactericidal activity of the composition of theinvention in all cases was significantly higher than that of thepreparation containing natural zinc in the form of similar salt. Thus itwas confirmed that the bacteriostatic and bactericidal activities of theantibacterial composition of the invention is directly attributable tothe presence of light isotope ⁶⁴Zn-enriched zinc in the form of a saltof an organic or inorganic acid.

Example 4

To confirm the effectiveness of the antibacterial composition of thepresent invention, a parallel experiment was carried out for itscomparison with known antibacterial agents. The following commercialantibacterial medications were used for the study: azithromycin andceftriaxone and norfloxacin. The initial concentration of 3000 mcg/mlwas used for the samples of all the above compounds. The conditions ofthe experiment were similar to those described in Example 1. Serialdilutions of the above antibacterial compounds were also prepared in thesame way as for the antibacterial composition of the invention. The samepathogenic strains of S. aureus ATCC 25923, E. coli ATCC 25922, P.aeruginosa ATCC 27853 were used for the study. The MIC and MBCestablished for the antibacterial agents are shown in Tables 9 and 10,respectively. The comparison of bactericidal and bacteriostaticactivities of the antibacterial composition of the present invention andcommercial antibacterial agents is shown in diagrams in FIG. 5.

TABLE 9 MIC of azithromycin, norfloxacin and ceftriaxone against teststrains MIC, MIC, MIC, mcg/ml mcg/ml mcg/ml Test cultures azithromycinnorfloxacin ceftriaxone S. aureus ATCC 25923 187.5 11.72* 11.72* E. coliATCC 25922 1500 23.44* 2.93* P. aeruginosa ATCC 27853 1500 187.5 750*Value is rounded off to the nearest 0.01.

TABLE 10 MBC of azithromycin, norfloxacin and ceftriaxone against teststrains MBC, MBC, MBC, mcg/ml mcg/ml mcg/ml Test cultures azithromycinnorfloxacin ceftriaxone S. aureus ATCC 25923 <1500 <1500 375 E. coliATCC 25922 <1500 <1500 11.72* P. aeruginosa ATCC 27853 <1500 <1500 <1500*Value is rounded off to the nearest 0.01.

As seen from the above data, the MIC and MBC of the commercialantibacterial agents are much inferior to those of the antibacterialcomposition of the invention which confirms its efficiency andexpediency of application for the control of pathogenic microorganisms.

Example 5

Determination of the Minimum Inhibitory Concentration ofDeuterium-Depleted Water

The minimum inhibitory concentration (MIC) of deuterium-depleted waterwas determined by serial dilutions in normal saline solution. Referencestrains of E. coli ATCC 25922 and P. aeruginosa ATCC 27859 were used astest cultures. For inoculation, microbial suspension equivalent to a 0.5standard McFarland (1.5×10⁸ cells/ml) was used. Then the dilutions withnormal saline solution were prepared so that the concentration ofmicroorganisms reached approximately 10⁴ cells/ml. The test cultureswere previously grown for 24 hours at 37° C. One-tenth of a milliliter(0.1 ml) of the suspension was placed into each test tube containing 4ml of the medium with the investigative active substance at a certainconcentration (without deuterium-depleted water in the control). Thetubes were incubated in an incubator at 37° C. for 24 hours. Afterincubation the inoculum from each tube was plated on Mueller-Hinton agarmedium for P. aeruginosa and on Endo for E. coli and incubated at 37° C.for another 24 hours. The results were evaluated based on the presenceof bacterial growth (turbidity) in the nutrient broth and, if necessary,by using other additional known bacteriological tests. The first fewtubes remained transparent due to the antimicrobial effect of the studysubstance. The emergence of growth in the other tubes suggests that thedrug concentration was below the minimum bactericidal concentration,which is determined by the last test tube in a series which had no signsof microbial growth. The results are shown in the table below andillustrated in FIGS. 5b and 5c .

TABLE 11 Antibacterial activity of deuterium-depleted water in theexperiments of Example 5 Strain of Volume of study substance in a tubeis 1 ml micro- Dilution of study substance: organism 1:5 1:10 1:20 1:401:80 1:160 1:320 1:640 1:1280 1:2560 E. coli ATCC − − − − − − − − − +25922 P. aeruginosa − − − − − − − − − + ATCC 27859

Findings

The basic solution of deuterium-depleted water without adding the studydrugs had a bactericidal effect on P. aeruginosa ATCC 27853 and E. coliATCC 25922 at concentrations of 10⁴, 10⁶, 10⁸ cells/ml.

-   -   Pure deuterium-depleted water is the best disinfectant for E.        coli ATCC 25922 and P. aeruginosa ATCC 27853 bacteria.

The MIC of deuterium-depleted water for P. aeruginosa ATCC and E. coliis its 1:1280 dilution.

1. An antibacterial composition comprising a bacteriostatic orbactericidal effective amount of ⁶⁴Zn_(e), present as an element or inthe form of a pharmaceutically acceptable salt, compound or complexthereof, wherein ⁶⁴Zn constitutes between about 90% and about 99.9% ofthe ⁶⁴Zn_(e), and further wherein the ⁶⁴Zn_(e) is present in at leastone salt form selected from a sulfate salt, glutamate salt, aspartatesalt, asparaginate salt, citrate salt, and ethylene diamine disuccinicacid (EDDS) salt.
 2. (canceled)
 3. The antibacterial composition ofclaim 1, further comprising at least one excipient.
 4. The compositionof claim 3, wherein the composition is formulated such that a singledose of the composition contains from about 11 mg to about 220 mg⁶⁴Zn_(e). 5-7. (canceled)
 8. The composition of claim 4, wherein theantibacterial composition is a tablet or capsule that contains betweenabout 10 mg and about 50 mg of ⁶⁴Zn_(e).
 9. An antibacterial compositioncomprising a bacteriostatic or bactericidal effective amount of⁶⁴Zn_(e), present as an element or in the form of a pharmaceuticallyacceptable salt, compound or complex thereof, wherein ⁶⁴Zn constitutesbetween about 90% and about 99.9% of the ⁶⁴Zn_(c), and, furthercomprising at least one excipient, wherein the composition is a tabletor capsule that contains between about 10 mg and about 50 mg of⁶⁴Zn_(e), and further wherein the ⁶⁴Zn_(e) is present in the form of⁶⁴Zn_(e) gluconate or ⁶⁴Zn_(e) bisglycinate chelate.
 10. The compositionof claim 8, wherein the ⁶⁴Zn is present in the form of one or morepharmaceutically acceptable salts selected from ⁶⁴Zn aspartate, ⁶⁴Znglutamate, ⁶⁴Zn EDDS, ⁶⁴Zn asparaginate, and ⁶⁴Zn sulfate.
 11. Thecomposition of claim 10 comprising about 15 mg, about 30 mg, or about 45mg ⁶⁴Zn_(e).
 12. A method of treating a condition that is caused by orcharacterized by a bacterial infection or excessive bacterial growth,wherein the method comprises administering an antibacterial compositionto a person or veterinary mammal that exhibits such a condition, whereinthe antibacterial composition comprises a bacteriostatic or bactericidaleffective amount of ⁶⁴Zn_(e), present as an element or in the form of apharmaceutically acceptable salt, compound or complex thereof, wherein⁶⁴Zn constitutes between about 90% and about 99.9% of the ⁶⁴Zn_(e), andfurther wherein the ⁶⁴Zn_(e) is present in at least one salt formselected from the sulfate salt, glutamate salt, aspartate salt,asparaginate salt, citrate salt, and ethylene diamine disuccinic acid(EDDS) salt.
 13. The method of claim 12, wherein the condition is acne.14. The method of claim 12, wherein the condition is sepsis.
 15. Themethod of claim 12, wherein the condition is a bacterial infection.